Quantitative determination of intracellular miRNA content using dual gold and iron nanoreporters and single particle ICP-ToF-MS.

MicroRNAs (miRNAs) are short single-stranded RNA sequences that play an important role in the initiation and progression of cancer. Therefore, the present work tries to establish an analytical platform for the quantitative determination of miRNA in cancer cell models without enzymatic amplification reactions. The developed assay is based on a sandwich double-hybridization reaction using a capture oligonucleotide conjugated to magnetic iron oxide microparticles and detecting oligonucleotide conjugated to a 40-nm gold nanoparticle, both particles coated with streptavidin. The optimization of the double-hybridization assay is conducted using inductively coupled plasma in single particle mode with a time-of-flight analyzer (SP-ICP-ToF-MS) for double detection of Au and Fe within the same event. The developed strategy was directly applied to the quantification of miR-16-5p in cell lysates without amplification reactions. For this aim, the cancer cell line of melanoma (A375) was studied, and two sample preparation strategies have been evaluated. Sequence capturing in extracted RNA provided best results allowing the determination at about 200 pM of miR-16-5p (for 2 × 10(6) cells). This strategy represents one of the few alternatives to obtain absolute quantification of miRNA in biological samples to permit the direct comparison among cell lines without amplification or transformation reactions of the original sequence.